CN111378614A - Single-layer compact milk cow mammary epithelial cell culture method - Google Patents

Single-layer compact milk cow mammary epithelial cell culture method Download PDF

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CN111378614A
CN111378614A CN202010193439.6A CN202010193439A CN111378614A CN 111378614 A CN111378614 A CN 111378614A CN 202010193439 A CN202010193439 A CN 202010193439A CN 111378614 A CN111378614 A CN 111378614A
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mammary epithelial
membrane
epithelial cells
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collagen
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CN111378614B (en
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李胜利
郑宇慧
刘钢
王蔚
曹志军
杨红建
王雅晶
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China Agricultural University
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Abstract

The invention provides a method for culturing single-layer compact milk cow mammary epithelial cells, which comprises the steps of paving collagen in a Transwell embedded sleeve chamber, and adjusting the culture medium composition and culture time of the milk cow mammary epithelial cells. Compared with the existing cell culture method, the method successfully constructs the mammary epithelial barrier model of the dairy cow, and can be widely applied to the aspects of researching the mechanism of mammary tissue damage when the dairy cow suffers from mastitis, milk substance transportation, the protection effect of different substances on the mammary epithelial barrier and the like.

Description

Single-layer compact milk cow mammary epithelial cell culture method
Technical Field
The invention relates to the technical field of biology, in particular to a method for culturing mammary epithelial cells of a single-layer compact milk cow.
Background
In the mammary gland of mammals, mammary epithelial cells are the first layer of cells on the surface layer of the mammary gland, and can not only secrete milk but also generate innate immune response to antigens such as escherichia coli.
Adjacent mammary epithelial cells are joined together by a cellular junction complex including Tight Junctions (TJ), Adhesive Junctions (AJ) and desmosomes (desmosomes). Wherein the connection complex, which is tightly connected to the topmost epithelial cells, is in a continuous band-like structure, so that adjacent epithelial cells are tightly connected to each other, and the paracellular flow of water molecules, ions, water and microorganisms is restricted, which is an important component of the blood-milk barrier. The desmosomes are tough and firm cell connecting structures, are connected with a network bracket formed by intermediate fibers to connect adjacent cells into a whole, can bear great tensile force, and have strong tensile force resistance, so that the connection between the cells is firmer.
Mammitis is a common and highly dangerous clinical disease in mammals, and cow mammary gland infected bacteria disturb the normal milk secretion function of mammary Epithelial cells of the cow, resulting in interruption of milk, acute mammitis often damages the blood milk barrier, and after the barrier function is destroyed, milk components such as α -lactalbumin and casein can enter blood, and blood components can also enter milk2Indicating the formation of a dense monolayer of cells.
Disclosure of Invention
The invention aims to provide a novel method for culturing single-layer compact milk cow mammary gland epithelial cells by utilizing milk cow mammary gland epithelial cells, and provides a compact single-layer milk cow mammary gland epithelial cell model for researching the mechanism of mammary gland tissue damage when a milk cow suffers from mastitis, milk substance transportation and the protection effect of different substances on a mammary gland epithelial barrier.
In order to realize the aim of the invention, the invention provides a method for culturing the single-layer compact milk cow mammary gland epithelial cells, which comprises the steps of inoculating the milk cow mammary gland epithelial cells into a Transwell nested perforated plate with a membrane, wherein collagen is paved on the upper chamber of the Transwell nested perforated plate, and then carrying out two-stage cell culture, namely culturing by adopting a culture medium 1 within 48 hours after inoculating the milk cow mammary gland epithelial cells, and then culturing by adopting a culture medium 2 within 72 hours.
Wherein the culture medium 1 is DMEM/F12 culture medium containing 10% fetal calf serum, 100U/mL penicillin, 100 mug/mL streptomycin, 15ng/mL human epidermal growth factor and 1% ITS. Wherein,% means volume percentage.
Medium 2 was DMEM/F12 medium containing 5% fetal bovine serum, 100U/mL penicillin, 100. mu.g/mL streptomycin and 1% ITS.
ITS is a solution containing 1g/L insulin, 0.55g/L transferrin, and 0.00067g/L sodium selenite, namely ITS solution (100 ×) available from Saimer Feishel technologies (China) Inc.
The method comprises the step of taking 10 percent of milk cow mammary epithelial cells4～105One/hole (preferably 2 × 10)4One/well) was inoculated in the upper chamber of a Transwell-embedded multiwell plate with a membrane in which collagen was laid, at 37 ℃ and 5% CO2Culturing under the condition.
In the above method, the culture medium was changed every 24 hours when the culture was carried out using the culture medium 1.
In the method, when the culture medium 2 is used for culture, the culture medium is replaced every 48 hours.
In the method, the concentration of the collagen is 0.012 mg/mL.
Preferably, the collagen is rat tail collagen (rat tail collagen), and the collagen solution for coating the multi-well plate is prepared with 0.36g/L acetic acid solution.
Preferably, the membrane-bearing Transwell nested multi-well plate is a 12-well plate.
The material of the membrane in the Transwell nested perforated plate with the membrane is a polyester membrane, the nested diameter is 6.5mm, and the growth area of the nested membrane is 0.33cm2The membrane had a pore size of 0.4 μm and a membrane thickness of 10 μm.
In the invention, the mammary epithelial cells of the dairy cow are inoculated in an upper chamber of a Transwell, 200 mu L of culture medium is added in the upper chamber, and 600 mu L of culture medium is added in a lower chamber; before cell inoculation, the upper and lower chambers of the Transwell need to be equilibrated by addition of cell culture medium 1.
In a second aspect, the present invention provides a monolayer of dense milk cow mammary epithelial cells cultured according to the above method as a barrier model of milk cow mammary epithelial cells.
Alternatively, the model uses TEER to assess the degree of compaction of the monolayer of cells. For example, after cell seeding, a single layer of cells is assayed for TEER at 48h intervals using a cell resistance apparatus (e.g., Millipore cell resistance apparatus MERS00002, USA) to 400. omega. cm2I.e. deemed satisfactory.
Optionally, the model uses Transmission Electron Microscopy (TEM) to detect desmosomal structures between milk cow mammary epithelial cells.
Alternatively, the model uses immunofluorescence staining to detect zonulin (ZO-1).
A schematic of the cell-cell adhesion complex in mammary epithelial cells is shown in figure 1. The technical scheme of the invention is shown in figure 2.
The model can be used for researching the damage mechanism of mammary tissue when the dairy cow suffers from mastitis, the transportation of milk substances, the protection effect of different substances on mammary epithelial barriers and the like.
The invention provides a novel culture method of milk cow mammary epithelial cells, and successfully constructs a milk cow mammary epithelial barrier model. According to the invention, collagen is laid in the upper chamber of the Transwell, and after cells are paved on the bottom of a culture dish, human epidermal growth factors in a culture medium are removed, the concentration of fetal calf serum is reduced, and a tight connection and desmosome structure is formed between mammary epithelial cells of a cow, so that an in-vitro cell model is provided for relevant experimental researches such as a mechanism of mammary tissue damage when the cow suffers from mastitis, milk substance transportation, a protective effect of different substances on a mammary epithelial barrier and the like.
Drawings
FIG. 1 is a schematic view of a cell-cell adhesion complex in mammary epithelial cells according to the present invention.
FIG. 2 is a technical scheme of the present invention.
FIG. 3 is a TEER variation diagram of a single layer of cow mammary gland epithelial cells in example 1 of the present invention.
FIG. 4 is a TEM image of desmosome structures among mammary epithelial cells of a cow in example 1 of the present invention. The circles in the figure indicate desmosome structures.
FIG. 5 is a photograph of immunofluorescence staining of Claudin ZO-1 in example 1 of the present invention.
FIG. 6 is a graph showing the changes of a monolayer of epithelial cells TEER before and after the Zhang treatment of Lactobacillus casei in example 2 of the present invention.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are commercially available products.
The ITS used in the following examples is a solution containing 1g/L insulin, 0.55g/L transferrin and 0.00067g/L sodium selenite. Namely ITS solution (100 x), available from Saimer Feishale technologies (China).
Rat tail collagen was purchased from siemer feishel technologies (china) ltd.
EXAMPLE 1 cow mammary epithelial cell culture method
The embodiment aims to provide a method for culturing mammary epithelial cells of a dairy cow, and provides a compact single-layer mammary epithelial cell model for deeply researching the mechanism of mammary tissue damage, milk substance transportation and the protection effect of different substances on a mammary epithelial barrier when the dairy cow suffers from mastitis.
The method comprises the following steps:
1. laying and balancing of Transwell collagen
(1) Preparation of 0.006mol/L (0.36g/L) acetic acid: 36 μ L of analytically pure acetic acid (glacial acetic acid) was taken and dissolved in 100mL of sterilized deionized water for further use.
(2) Preparation of 0.012mg/mL collagen: mu.L of rat tail collagen (3.52mg/mL) was dissolved in 0.006mol/L of acetic acid as described above.
(3) 50. mu.L of the above 0.012mg/mL collagen was uniformly applied to a Transwell nested chamber (membrane growth area 0.33 cm)2) And placed in an incubator at 37 ℃ overnight.
(4) At least 1h before use, 600 mu L of culture solution is added into a 12-hole plate, then the nest paved with collagen is placed, and 200 mu L of culture solution is added into the nest.
2. Construction of single-layer dairy cow mammary epithelial cell barrier model
(1) When Bovine Mammary Epithelial Cells (BMECS) were plated around 80% of the cell culture dish, 1mL of 0.05% trypsin (containing 0.02% EDTA) was added for digestion, and when 70% of the cells were rounded and shrunk under an inverted microscope, 3mL of the medium was added to stop digestion when they fell off the bottom of the dish.
(2) Collecting cells, centrifuging at 1000rpm for 1min 30s, discarding supernatant, adding 1mL culture medium to prepare cell suspension, mixing with 0.4% trypan blue dye solution according to the ratio of 1: 1, 20. mu.L of the mixed solution was aspirated by a pipette gun, and gradually dropped along the edge of the counting plate to fill the counting plate, followed by counting with a cell counter (TC10 automatic cell counter, Bio-Rad).
(3) According to the cell counting result, the mammary epithelial cells of the milk cow are counted as 2 × 104The individual/Transwell cells were inoculated into a Transwell nest with collagen laid and equilibrated in an incubator and placed at 37 ℃ in 5% CO2Culturing in a constant temperature incubator.
(4) The cells of (3) were cultured in DMEM/F12 medium (Medium 1) containing 10% fetal bovine serum, 100U/mL penicillin, 100. mu.g/mL streptomycin, 15ng/mL human epidermal growth factor, 1% ITS (insulin-transferrin-selenium-ethanolamine solution), with medium changes every 24 h.
(5) After culturing the cells for 48h as above, the medium was replaced with a new one (medium 2): DMEM/F12 medium containing 5% fetal bovine serum, 100U/mL penicillin, 100. mu.g/mL streptomycin, 1% ITS. The medium was changed every 48 h.
(6) After the milk cow mammary gland epithelial cells were cultured to the medium in (4), TEER of the monolayer cells was measured every 48h using Millipore cell resistance meter MERS 00002.
(7) The tight junction protein ZO-1 in the mammary epithelial barrier of the milk cow is detected by the TEER determination (figure 3), the TEM observation of intercellular desmosome structure (figure 4) and the immunofluorescence staining method (figure 5), which shows that the mammary epithelial cells of the milk cow form desmosomes and tight junctions, and the construction of the mammary epithelial barrier model of the milk cow is successful.
Example 2 screening of substances for enhancing mammary epithelial barrier function Using model
This example aims to provide a basis for the prevention and treatment of mastitis in dairy cows by screening a substance that enhances the barrier function of mammary epithelial cells using the method for culturing mammary epithelial cells in dairy cows described in example 1.
The method comprises the following steps:
1. laying and balancing of Transwell collagen
(1) Preparation of 0.006mol/L (0.36g/L) acetic acid: 36 μ L of analytically pure acetic acid (glacial acetic acid) was taken and dissolved in 100mL of sterilized deionized water for further use.
(2) Preparation of 0.012mg/mL collagen: mu.L of rat tail collagen (3.52mg/mL) was dissolved in 0.006mol/L of acetic acid as described above.
(3) 50. mu.L of the above 0.012mg/mL collagen was uniformly applied to a Transwell nested chamber (membrane growth area 0.33 cm)2) And placed in an incubator at 37 ℃ overnight.
(4) At least 1h before use, 600 mu L of culture solution is added into a 12-hole plate, then the nest paved with collagen is placed, and 200 mu L of culture solution is added into the nest.
2. Screening of substance for enhancing mammary epithelial barrier function
(1) When Bovine Mammary Epithelial Cells (BMECS) were plated around 80% of the cell culture dish, 1mL of 0.05% trypsin (containing 0.02% EDTA) was added for digestion, and when 70% of the cells were rounded and shrunk under an inverted microscope, 3mL of the medium was added to stop digestion when they fell off the bottom of the dish.
(2) Collecting cells, centrifuging at 1000rpm for 1min 30s, discarding supernatant, adding 1mL culture medium to prepare cell suspension, mixing with 0.4% trypan blue dye solution according to the ratio of 1: 1, 20. mu.L of the mixed solution was aspirated by a pipette gun, and gradually dropped along the edge of the counting plate to fill the counting plate, followed by counting with a cell counter (TC10 automatic cell counter, Bio-Rad).
(3)According to the cell counting result, the mammary epithelial cells of the milk cow are counted as 2 × 104The individual/Transwell cells were inoculated into a well-balanced Transwell nested chamber with collagen laid in an incubator and placed at 37 ℃ in 5% CO2Culturing in a constant temperature incubator.
(4) The cells of (3) were cultured in DMEM/F12 medium (Medium 1) containing 10% fetal bovine serum, 100U/mL penicillin, 100. mu.g/mL streptomycin, 15ng/mL human epidermal growth factor, 1% ITS (insulin-transferrin-selenium-ethanolamine solution), with medium changes every 24 h.
(5) After culturing the cells for 48h as above, the medium was replaced with a new one (medium 2): DMEM/F12 medium containing 5% fetal bovine serum, 100U/mL penicillin, 100. mu.g/mL streptomycin, 1% ITS. The medium was changed every 48 h.
(6) After the milk cow mammary gland epithelial cells were cultured to the medium in (4), TEER of the monolayer cells was measured every 48h using Millipore cell resistance meter MERS 00002.
(7) When the TEER reaches 400 omega cm2Adding Lactobacillus casei Zhang, and standing at 37 deg.C and 5% CO2TEER was determined after 3h incubation in a thermostated incubator (figure 6).
Determination by TEER found 105The method has the advantages that the TEER of the mammary epithelial cells of the single-layer compact milk cow can be remarkably increased by processing the CFU Lactobacillus casei Zhang (provided by a key laboratory of the department of dairy biotechnology and engineering education of the inner Mongolia university) for 3 hours, namely, the integrity and the compactness of the barrier can be effectively enhanced.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Claims (10)

1. The method for culturing the single-layer compact dairy cow mammary epithelial cells is characterized in that the dairy cow mammary epithelial cells are inoculated into a Transwell nested perforated plate with a membrane, wherein collagen is laid in the upper chamber of the Transwell nested perforated plate, and then two-stage cell culture is carried out, namely, after the dairy cow mammary epithelial cells are inoculated, a culture medium 1 is adopted for culture within 48 hours, and then a culture medium 2 is adopted for culture within 72 hours;
wherein the culture medium 1 is DMEM/F12 culture medium containing 10% fetal calf serum, 100U/mL penicillin, 100 mug/mL streptomycin, 15ng/mL human epidermal growth factor and 1% ITS;
the culture medium 2 is DMEM/F12 culture medium containing 5% fetal calf serum, 100U/mL penicillin, 100. mu.g/mL streptomycin and 1% ITS;
the ITS is a solution containing 1g/L insulin, 0.55g/L transferrin and 0.00067g/L sodium selenite.
2. The method of claim 1, wherein the cow mammary gland epithelial cells are treated at 10 ° f4～105The cells/well were inoculated into the upper chamber of a Transwell-embedded multiwell plate with a membrane, in which collagen was laid, at 37 ℃ and 5% CO2Culturing under the condition.
3. The method according to claim 1, wherein the culture is carried out using medium 1, and the medium is changed every 24 hours.
4. The method according to claim 1, wherein the culture is carried out using medium 2, and the medium is changed every 48 hours.
5. The method of claim 1, wherein the collagen concentration is 0.012 mg/mL.
6. The method according to any one of claims 1 to 5, wherein the collagen is rat tail collagen, and the collagen solution used for laying the porous plate is prepared with 0.36g/L acetic acid solution.
7. The method of any one of claims 1 to 5, wherein the membrane-bearing Transwell nested multi-well plate is a 12-well plate.
8. The method as claimed in any one of claims 1 to 5, wherein the material of the membrane in the Transwell-equipped nested perforated plate with membrane is polyester membrane, the nested diameter is 6.5mm, and the growth area of the nested membrane is 0.33cm2The membrane had a pore size of 0.4 μm and a membrane thickness of 10 μm.
9. The monolayer dense dairy cow mammary epithelial cells obtained by culturing according to the method of any one of claims 1 to 8.
10. The use of the dense single-layered cow mammary epithelial cells of claim 9 in any one of the following applications:
1) the method is used for researching the destruction mechanism of mammary tissue when the dairy cow suffers from mastitis;
2) the method is used for the research on the transportation of milk substances of cows and the protective effect of different substances on mammary epithelial barriers.
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111454879A (en) * 2020-03-23 2020-07-28 中国农业大学 Construction method of mammary epithelial cell model of escherichia coli infected single-layer compact dairy cow

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CN108396007A (en) * 2018-03-15 2018-08-14 东北农业大学 The method of external structure milk ox blood breast barrier threedimensional model

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111454879A (en) * 2020-03-23 2020-07-28 中国农业大学 Construction method of mammary epithelial cell model of escherichia coli infected single-layer compact dairy cow

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